Thyroid hormone receptors form distinct nuclear protein-dependent and independent complexes with a thyroid hormone response element.
We have examined the binding of nuclear proteins and recombinant thyroid hormone receptors (TRs) to the palindromic thyroid hormone responsive element AGGTCATGACCT (TREp) using a gel electrophoretic mobility shift assay. Four specific protein-DNA complexes were detected after incubation of nuclear extracts (NE) from T3-responsive pituitary (GH3) cells with a TREp-containing DNA fragment. This was compared with the TREp binding of reticulocyte lysate-synthesized TRs. TR alpha 1 and TR beta 2 each formed a single major TR:TREp complex which comigrated with the least retarded complex formed by GH3 NE, while TR beta 1 formed multiple complexes suggesting that it can bind to TREp as an oligomer. Interestingly, coincubation of 35S-TR alpha 1, GH3 NE, and unlabeled TREp resulted in not only the 35S-TR:TREp complex, but in two additional more greatly retarded complexes containing 35S-TR alpha 1 and comigrating with those formed by GH3 extract alone. Incubation of each of the TRs with NE from COS-7 cells, which do not possess sufficient endogenous TRs to mediate T3-responses, resulted in formation of a new, more greatly shifted complex. A similar, heat labile activity which altered mobility of the TR:TRE complex was also present in NE from T3-unresponsive JEG-3 cells. At high concentration of NE, all of the TR bound to TREp was more greatly retarded than in the absence of NE. Truncation of TR alpha 1 at amino acid 210 prevented additional complex formation in the presence of NE without affecting DNA binding, suggesting that the carboxyl-terminus of the TRs is essential for interaction with nuclear proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Lazar
M A
MA
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104.
Berrodin
T J
TJ
D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D010902:Pituitary Gland / Q000737:chemistry; D012333:RNA, Messenger / Q000096:biosynthesis; D011988:Receptors, Thyroid Hormone / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid; D012156:Reticulocytes / Q000378:metabolism; D013462:Sulfur Radioisotopes
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D011988:Receptors, Thyroid Hormone; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D004247:DNA